Activin-actrii antagonists and uses for increasing red blood cell levels

ABSTRACT

In certain aspects, the present invention provides compositions and methods for increasing red blood cell and/or hemoglobin levels in vertebrates, including rodents and primates, and particularly in humans.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/240,276, filed Jan. 4, 2019, which is a continuation of U.S.application Ser. No. 13/189,353, filed Jul. 22, 2011 (now abandoned),which is a continuation of U.S. application Ser. No. 12/286,333, filedSep. 29, 2008 (now U.S. Pat. No. 8,007,809), which is a continuation ofU.S. application Ser. No. 12/002,872, filed Dec. 18, 2007 (now U.S. Pat.No. 7,988,973), which claims the benefit of U.S. Provisional PatentApplication No. 60/875,682, filed Dec. 18, 2006 (now expired). Thedisclosures of each of the foregoing applications are herebyincorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Dec. 22, 2020, is named1848179-0002-022-106_Seq.txt, and is 39,570 bytes in size.

BACKGROUND OF THE INVENTION

The mature red blood cell, or erythrocyte, is responsible for oxygentransport in the circulatory systems of vertebrates. Red blood cellscarry high concentrations of hemoglobin, a protein that binds oxygen inthe lungs at relatively high partial pressure of oxygen (pO₂) anddelivers oxygen to areas of the body with a relatively low pO₂.

Mature red blood cells are produced from pluripotent hematopoietic stemcells in a process termed erythropoiesis. In post-natal individuals,erythropoiesis occurs primarily in the bone marrow and in the red pulpof the spleen. The coordinated action of various signaling pathwayscontrol the balance of cell proliferation, differentiation, survival anddeath. Under normal conditions, red blood cells are produced at a ratethat maintains a constant red cell mass in the body, and production mayincrease or decrease in response to various stimuli, including increasedor decreased oxygen tension or tissue demand. The process oferythropoiesis begins with the formation of lineage committed precursorcells and proceeds through a series of distinct precursor cell types.The final stages of erythropoiesis occur as reticulocytes are releasedinto the bloodstream and lose their mitochondria and ribosomes whileassuming the morphology of mature red blood cell. An elevated level ofreticulocytes, or an elevated reticulocyte:erythrocyte ratio, in theblood is indicative of increased red blood cell production rates.

Erythropoietin (Epo) is widely recognized as the most significantpositive regulator of erythropoiesis in post-natal vertebrates. Eporegulates the compensatory erythropoietic response to reduced tissueoxygen tension (hypoxia) and low red blood cell levels or low hemoglobinlevels. In humans, elevated Epo levels promote red blood cell formationby stimulating the generation of erythroid progenitors in the bonemarrow and spleen. In the mouse, Epo enhances erythropoiesis primarilyin the spleen.

Various forms of recombinant Epo are used by physicians to increase redblood cell levels in a variety of clinical settings, and particularlyfor the treatment of anemia. Anemia is a broadly-defined conditioncharacterized by lower than normal levels of hemoglobin or red bloodcells in the blood. In some instances, anemia is caused by a primarydisorder in the production or survival of red blood cells. Morecommonly, anemia is secondary to diseases of other systems (Weatherall &Provan (2000) Lancet 355, 1169-1175). Anemia may result from a reducedrate of production or increased rate of destruction of red blood cellsor by loss of red blood cells due to bleeding. Anemia may result from avariety of disorders that include, for example, chronic renal failure,myelodysplastic syndrome, rheumatoid arthritis, and bone marrowtransplantation.

Treatment with Epo typically causes a rise in hemoglobins by about 1-3g/dL in healthy humans over a period of weeks. When administered toanemic individuals, this treatment regimen often provides substantialincreases in hemoglobin and red blood cell levels and leads toimprovements in quality of life and prolonged survival. Epo is notuniformly effective, and many individuals are refractory to even highdoses (Horl et al. (2000) Nephrol Dial Transplant 15, 43-50). Over 50%of patients with cancer have an inadequate response to Epo,approximately 10% with end-stage renal disease are hyporesponsive(Glaspy et al. (1997) J Clin Oncol 15, 1218-1234; Demetri et al. (1998)J Clin Oncol 16, 3412-3425), and less than 10% with myelodysplasticsyndrome respond favorably (Estey (2003) Curr Opin Hematol 10, 60-67).Several factors, including inflammation, iron and vitamin deficiency,inadequate dialysis, aluminum toxicity, and hyperparathyroidism maypredict a poor therapeutic response, the molecular mechanisms ofresistance to Epo are as yet unclear.

Thus, it is an object of the present disclosure to provide alternativecompositions and methods for increasing red blood cell levels inpatients.

SUMMARY OF THE INVENTION

In part, the disclosure demonstrates that activin antagonists, as wellas ActRIIa and ActRIIb antagonists, can be used to increase red bloodcell and hemoglobin levels. In particular, the disclosure demonstratesthat a soluble form of ActRIIa acts as an inhibitor of activin and, whenadministered in vivo, increases red blood cell levels in the blood. Amilder effect was observed with a soluble form of ActRIIb, which bindsActivin A with lesser affinity than soluble ActRIIa. While solubleActRIIa and ActRIIb may affect red blood cell levels through a mechanismother than activin antagonism, the disclosure nonetheless demonstratesthat desirable therapeutic agents may be selected on the basis ofactivin antagonism or ActRII antagonism or both. Such agents arereferred to collectively as activin-ActRII antagonists. Therefore, incertain embodiments, the disclosure provides methods for usingactivin-ActRII antagonists, including, for example, activin-bindingActRIIa polypeptides, activin-binding ActRIb polypeptides, anti-activinantibodies, anti-ActRIIa antibodies, anti-ActRIb antibodies, activin-,ActRIIb-, or ActRIIa-targeted small molecules and aptamers, and nucleicacids that decrease expression of activin, ActRIIb, or ActRIIa, toincrease red blood cell and hemoglobin levels in patients and to treatdisorders associated with low red blood cell or hemoglobin levels inpatients in need thereof. As described in U.S. patent application Ser.No. 11/603,485, incorporated by reference herein, activin-ActRIIaantagonists can be used to promote bone growth and increase bonedensity. As described herein, the effects of such antagonists on redblood cell levels are more rapid and occur at lower doses than theeffects of such antagonists on bone. Thus, in certain embodiments, thedisclosure provides methods for using an activin-ActRIIa antagonist toincrease red blood cell or hemoglobin levels without causing asignificant increase in bone density. For example, a method may causeless than 3%, 5%, 10% or 15% increase in bone density. This selectiveeffect may be achieved by using, for example, lower doses ofactivin-ActRIIa antagonist, less frequent doses, or by using anactivin-ActRIIa antagonist with a shorter serum half-life at doses andfrequencies calculated to provide a lower serum concentration.

In certain aspects, the disclosure provides polypeptides comprising asoluble, activin-binding ActRII polypeptide that binds to activin. Theactivin binding polypeptide may be an ActRIIa polypeptide or an ActRIIbpolypeptide. ActRII polypeptides may be formulated as a pharmaceuticalpreparation comprising the activin-binding ActRII polypeptide and apharmaceutically acceptable carrier. The activin-binding ActRIIpolypeptide may bind to activin with a K_(D) less than 1 micromolar orless than 100, 10 or 1 nanomolar. Optionally, the activin-binding ActRIIpolypeptide selectively binds activin versus GDF11 and/or GDF8, andoptionally with a K_(D) that is at least 10-fold, 20-fold or 50-foldlower with respect to activin than with respect to GDF11 and/or GDF8.While not wishing to be bound to a particular mechanism of action, it isexpected that this degree of selectivity for activin inhibition overGDF11/GDF8 inhibition accounts for effects on bone or erythropoiesiswithout a consistently measurable effect on muscle. In many embodiments,an ActRII polypeptide will be selected for causing less than 15%, lessthan 10% or less than 5% increase in muscle at doses that achievedesirable effects on red blood cell levels. The composition may be atleast 95% pure, with respect to other polypeptide components, asassessed by size exclusion chromatography, and optionally, thecomposition is at least 98% pure. An activin-binding ActRIIa polypeptidefor use in such a preparation may be any of those disclosed herein, suchas a polypeptide having an amino acid sequence selected from SEQ ID NOs:2, 3, 7 or 12, or having an amino acid sequence that is at least 80%,85%, 90%, 95%, 97% or 99% identical to an amino acid sequence selectedfrom SEQ ID NOs: 2, 3, 7, 12 or 13. An activin-binding ActRIIapolypeptide may include a functional fragment of a natural ActRIIapolypeptide, such as one comprising at least 10, 20 or 30 amino acids ofa sequence selected from SEQ ID NOs: 1-3 or a sequence of SEQ ID NO: 2,lacking the C-terminal 10 to 15 amino acids (the “tail”). Anactivin-binding ActRIb polypeptide for use in such a preparation may beany of those disclosed herein, such as a polypeptide having an aminoacid sequence selected from SEQ ID NOs: 16, 17, 20, or 21 or having anamino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99%identical to an amino acid sequence selected from SEQ ID NOs: 16, 17,20, or 21. An activin-binding ActRIIb polypeptide may include afunctional fragment of a natural ActRIIb polypeptide, such as onecomprising at least 10, 20 or 30 amino acids of SEQ ID NOs: 15-17 or asequence lacking the C-terminal 10 to 15 amino acids (the “tail”) suchas SEQ ID NO: 17.

A soluble, activin-binding ActRII polypeptide may include one or morealterations in the amino acid sequence (e.g., in the ligand-bindingdomain) relative to a naturally occurring ActRII polypeptide. Examplesof altered ActRIIa and ActRIIb polypeptides are provided in WO2006/012627, pp. 59-60 and pp. 55-58, respectively, which isincorporated by reference herein. The alteration in the amino acidsequence may, for example, alter glycosylation of the polypeptide whenproduced in a mammalian, insect or other eukaryotic cell or alterproteolytic cleavage of the polypeptide relative to the naturallyoccurring ActRII polypeptide.

An activin-binding ActRII polypeptide may be a fusion protein that has,as one domain, an ActRII polypeptide, (e.g., a ligand-binding portion ofan ActRIIa or ActRIIb) and one or more additional domains that provide adesirable property, such as improved pharmacokinetics, easierpurification, targeting to particular tissues, etc. For example, adomain of a fusion protein may enhance one or more of in vivo stability,in vivo half life, uptake/administration, tissue localization ordistribution, formation of protein complexes, multimerization of thefusion protein, and/or purification. An activin-binding ActRII fusionprotein may include an immunoglobulin Fc domain (wild-type or mutant) ora serum albumin or other polypeptide portion that provides desirableproperties such as improved pharmacokinetics, improved solubility orimproved stability. In a preferred embodiment, an ActRII-Fc fusioncomprises a relatively unstructured linker positioned between the Fcdomain and the extracellular ActRII domain. This unstructured linker maycorrespond to the roughly 15 amino acid unstructured region at theC-terminal end of the extracellular domain of ActRII (the “tail”), or itmay be an artificial sequence of 1, 2, 3, 4 or 5 amino acids or a lengthof between 5 and 15, 20, 30, 50 or more amino acids that are relativelyfree of secondary structure, or a mixture of both. A linker may be richin glycine and proline residues and may, for example, contain a singlesequence of threonine/serine and glycines or repeating sequences ofthreonine/serine and glycines (e.g., TG₄ (SEQ ID NO: 22) or SG₄ (SEQ IDNO: 23) singlets or repeats). A fusion protein may include apurification subsequence, such as an epitope tag, a FLAG tag, apolyhistidine sequence, and a GST fusion. Optionally, a soluble ActRIIpolypeptide includes one or more modified amino acid residues selectedfrom: a glycosylated amino acid, a PEGylated amino acid, a farnesylatedamino acid, an acetylated amino acid, a biotinylated amino acid, anamino acid conjugated to a lipid moiety, and an amino acid conjugated toan organic derivatizing agent. A pharmaceutical preparation may alsoinclude one or more additional compounds such as a compound that is usedto treat a bone disorder. Preferably, a pharmaceutical preparation issubstantially pyrogen free. In general, it is preferable that an ActRIIprotein be expressed in a mammalian cell line that mediates suitablynatural glycosylation of the ActRII protein so as to diminish thelikelihood of an unfavorable immune response in a patient. Human and CHOcell lines have been used successfully, and it is expected that othercommon mammalian expression systems will be useful.

As described herein, ActRIIa proteins designated ActRIIa-Fc (a form witha minimal linker between the ActRIIa portion and the Fc portion) havedesirable properties, including selective binding to activin versus GDF8and/or GDF11, high affinity ligand binding and serum half life greaterthan two weeks in animal models. In certain embodiments the inventionprovides ActRII-Fc polypeptides and pharmaceutical preparationscomprising such polypeptides and a pharmaceutically acceptableexcipient.

In certain aspects, the disclosure provides nucleic acids encoding asoluble activin-binding ActRII polypeptide, such as an ActRIIa orActRIIb polypeptide. An isolated polynucleotide may comprise a codingsequence for a soluble, activin-binding ActRII polypeptide, such asdescribed above. For example, an isolated nucleic acid may include asequence coding for an extracellular domain (e.g., ligand-bindingdomain) of an ActRII and a sequence that would code for part or all ofthe transmembrane domain and/or the cytoplasmic domain of an ActRII, butfor a stop codon positioned within the transmembrane domain or thecytoplasmic domain, or positioned between the extracellular domain andthe transmembrane domain or cytoplasmic domain. For example, an isolatedpolynucleotide may comprise a full-length ActRIIa polynucleotidesequence such as SEQ ID NO: 4 or 5 or a full-length ActRIIbpolynucleotide sequence such as SEQ ID NO: 18, or a partially truncatedversion of ActRIIa or ActRIIb, said isolated polynucleotide furthercomprising a transcription termination codon at least six hundrednucleotides before the 3′-terminus or otherwise positioned such thattranslation of the polynucleotide gives rise to an extracellular domainoptionally fused to a truncated portion of a full-length ActRII. Apreferred nucleic acid sequence for ActRIIa is SEQ ID NO:14. Nucleicacids disclosed herein may be operably linked to a promoter forexpression, and the disclosure provides cells transformed with suchrecombinant polynucleotides. Preferably the cell is a mammalian cellsuch as a CHO cell.

In certain aspects, the disclosure provides methods for making asoluble, activin-binding ActRII polypeptide. Such a method may includeexpressing any of the nucleic acids (e.g., SEQ ID NO: 4, 5 14, 18, or19) disclosed herein in a suitable cell, such as a Chinese hamster ovary(CHO) cell. Such a method may comprise: a) culturing a cell underconditions suitable for expression of the soluble ActRII polypeptide,wherein said cell is transformed with a soluble ActRII expressionconstruct; and b) recovering the soluble ActRII polypeptide soexpressed. Soluble ActRII polypeptides may be recovered as crude,partially purified or highly purified fractions. Purification may beachieved by a series of purification steps, including, for example, one,two or three or more of the following, in any order: protein Achromatography, anion exchange chromatography (e.g., Q sepharose),hydrophobic interaction chromatography (e.g., phenylsepharose), sizeexclusion chromatography, and cation exchange chromatography.

In certain aspects, an activin-ActRII antagonist disclosed herein, suchas a soluble, activin-binding ActRIIa polypeptide or soluble,activin-binding ActRIIb polypeptide, may be used in a method forpromoting red blood cell production or increasing red blood cell levelsin a subject. In certain embodiments, the disclosure provides methodsfor treating a disorder associated with low red blood cell counts or lowhemoglobin levels (e.g., an anemia), or to promote red blood cellproduction, in patients in need thereof. A method may compriseadministering to a subject in need thereof an effective amount ofactivin-ActRII antagonist. In certain aspects, the disclosure providesuses of activin-ActRII antagonists for making a medicament for thetreatment of a disorder or condition as described herein.

In certain aspects, the disclosure provides a method for identifying anagent that stimulates production of red blood cells. The methodcomprises: a) identifying a test agent that binds to activin or aligand-binding domain of an ActRII polypeptide; and b) evaluating theeffect of the agent on the levels of red blood cells, hemoglobin, and/orred blood cell precursor levels (e.g., reticulocyte levels).

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or patent application filed contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the office uponrequest and payment of the necessary fee.

FIG. 1A and FIG. 1B show the purification of ActRIIa-hFc expressed inCHO cells. The protein purifies as a single, well-defined peak asvisualized by sizing column (FIG. 1A) and Coomassie stained SDS-PAGE(FIG. 1B) (left lane: molecular weight standards; right lane:ActRIIa-hFc).

FIG. 2A and FIG. 2B show the binding of ActRIIa-hFc to activin (FIG. 2A)and GDF-11 (FIG. 2B), as measured by BiaCore™ assay.

FIG. 3A and FIG. 3B show the effects of ActRIIa-hFc on red blood cellcounts in female non-human primates. Female cynomolgus monkeys (fourgroups of five monkeys each) were treated with placebo or 1 mg/kg, 10mg/kg or 30 mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day 21.FIG. 3A shows red blood cell (RBC) counts. FIG. 3B shows hemoglobinlevels. Statistical significance is relative to baseline for eachtreatment group. At day 57, two monkeys remained in each group.

FIG. 4A and FIG. 4B show the effects of ActRIIa-hFc on red blood cellcounts in male non-human primates. Male cynomolgus monkeys (four groupsof five monkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg or30 mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day 21. FIG. 4Ashows red blood cell (RBC) counts. FIG. 4B shows hemoglobin levels.Statistical significance is relative to baseline for each treatmentgroup. At day 57, two monkeys remained in each group.

FIG. 5A and FIG. 5B show the effects of ActRIIa-hFc on reticulocytecounts in female non-human primates. Cynomolgus monkeys (four groups offive monkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg or 30mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day 21. FIG. 5A showsabsolute reticulocyte counts. FIG. 5B shows the percentage ofreticulocytes relative to RBCs. Statistical significance is relative tobaseline for each group. At day 57, two monkeys remained in each group.

FIG. 6A and FIG. 6B shows the effects of ActRIIa-hFc on reticulocytecounts in female non-human primates. Cynomolgus monkeys (four groups offive monkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg or 30mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day 21. FIG. 6A showsabsolute reticulocyte counts. FIG. 6B shows the percentage ofreticulocytes relative to RBCs. Statistical significance is relative tobaseline for each group. At day 57, two monkeys remained in each group.

FIG. 7 shows results from the human clinical trial described in Example5, where the area-under-curve (AUC) and administered dose of ActRIIa-hFchave a linear correlation, regardless of whether ActRIIa-hFc wasadministered intravenously (IV) or subcutaneously (SC).

FIG. 8 shows a comparison of serum levels of ActRIIa-hFc in patientsadministered IV or SC.

FIG. 9 shows bone alkaline phosphatase (BAP) levels in response todifferent dose levels of ActRIIa-hFc. BAP is a marker for anabolic bonegrowth.

FIG. 10 depicts the median change from baseline of hematocrit levelsfrom the human clinical trial described in Example 5. ActRIIa-hFc wasadministered intravenously (IV) at the indicated dosage.

FIG. 11 depicts the median change from baseline of hemoglobin levelsfrom the human clinical trial described in Example 5. ActRIIa-hFc wasadministered intravenously (IV) at the indicated dosage.

FIG. 12 depicts the median change from baseline of RBC (red blood cell)count from the human clinical trial described in Example 5. ActRIIa-hFcwas administered intravenously (IV) at the indicated dosage.

FIG. 13 depicts the median change from baseline of reticulocyte countfrom the human clinical trial described in Example 5. ActRIIa-hFc wasadministered intravenously (IV) at the indicated dosage.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. The family is dividedinto two general branches: the BMP/GDF and the TGF-beta/Activin/BMP10branches, whose members have diverse, often complementary effects. Bymanipulating the activity of a member of the TGF-beta family, it isoften possible to cause significant physiological changes in anorganism. For example, the Piedmontese and Belgian Blue cattle breedscarry a loss-of-function mutation in the GDF8 (also called myostatin)gene that causes a marked increase in muscle mass. Grobet et al., NatGenet. 1997, 17(1):71-4. Furthermore, in humans, inactive alleles ofGDF8 are associated with increased muscle mass and, reportedly,exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

Activins are dimeric polypeptide growth factors that belong to theTGF-beta superfamily. There are three principal activin forms (A, B, andAB) that are homo/heterodimers of two closely related β subunits(β_(A)β_(A), β_(B)β_(B), and β_(A)β_(B), respectively). The human genomealso encodes an activin C and an activin E, which are primarilyexpressed in the liver, and heterodimeric forms containing fc or PE arealso known. In the TGF-beta superfamily, activins are unique andmultifunctional factors that can stimulate hormone production in ovarianand placental cells, support neuronal cell survival, influencecell-cycle progress positively or negatively depending on cell type, andinduce mesodermal differentiation at least in amphibian embryos (DePaoloet al., 1991, Proc Soc Ep Biol Med. 198:500-512; Dyson et al., 1997,Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol. 55:953-963).Moreover, erythroid differentiation factor (EDF) isolated from thestimulated human monocytic leukemic cells was found to be identical toactivin A (Murata et al., 1988, PNAS, 85:2434). It has been suggestedthat activin A promotes erythropoiesis in the bone marrow. In severaltissues, activin signaling is antagonized by its related heterodimer,inhibin. For example, during the release of follicle-stimulating hormone(FSH) from the pituitary, activin promotes FSH secretion and synthesis,while inhibin prevents FSH secretion and synthesis. Other proteins thatmay regulate activin bioactivity and/or bind to activin includefollistatin (FS), follistatin-related protein (FSRP) andα₂-macroglobulin.

TGF-β signals are mediated by heteromeric complexes of type I and typeII serine/threonine kinase receptors, which phosphorylate and activatedownstream Smad proteins upon ligand stimulation (Massagué, 2000, Nat.Rev. Mol. Cell Biol. 1:169-178). These type I and type II receptors aretransmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine specificity. Type Ireceptors are essential for signaling; and type II receptors arerequired for binding ligands and for expression of type I receptors.Type I and II activin receptors form a stable complex after ligandbinding, resulting in phosphorylation of type I receptors by type IIreceptors.

Two related type II receptors (ActRII), ActRIIa and ActRIIb, have beenidentified as the type II receptors for activins (Mathews and Vale,1991, Cell 65:973-982; Attisano et al., 1992, Cell 68: 97-108). Besidesactivins, ActRIIa and ActRIb can biochemically interact with severalother TGF-β family proteins, including BMP7, Nodal, GDF8, and GDF11(Yamashita et al., 1995, J. Cell Biol. 130:217-226; Lee and McPherron,2001, Proc. Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol.Cell 7: 949-957; Oh et al., 2002, Genes Dev. 16:2749-54). ALK4 is theprimary type I receptor for activins, particularly for activin A, andALK-7 may serve as a receptor for activins as well, particularly foractivin B.

As demonstrated herein, a soluble ActRIIa polypeptide (sActRIIa), whichshows substantial preference in binding to activin A as opposed to otherTGF-beta family members, such as GDF8 or GDF11, is effective to increasered blood cell levels in vivo. While not wishing to be bound to anyparticular mechanism, it is expected that the effect of sActRIIa iscaused primarily by an activin antagonist effect, given the very strongactivin binding (picomolar dissociation constant) exhibited by theparticular sActRIIa construct used in these studies. Regardless ofmechanism, it is apparent from this disclosure that ActRIIa-activinantagonists increase red blood cell levels in rodents, monkeys andhumans. It should be noted that hematopoiesis is a complex process,regulated by a variety of factors, including erythropoietin, G-CSF andiron homeostasis. The terms “increase red blood cell levels” and“promote red blood cell formation” refer to clinically observablemetrics, such as hematocrit, red blood cell counts and hemoglobinmeasurements, and are intended to be neutral as to the mechanism bywhich such changes occur.

As also demonstrated herein, a soluble ActRIIb polypeptide (sActRIIb) iseffective to increase reticulocyte levels in vivo, an effect which, overa longer time period is expected to cause increased hemotocrit levels.

The data reported herein with respect to non-human primates arereproducible in mice, rats and humans as well, and therefore, thisdisclosure provides methods for using ActRII polypeptides and otheractivin-ActRII antagonists to promote red blood cell production andincrease red blood cell levels in mammals ranging from rodents tohumans. Activin-ActRII antagonists include, for example, activin-bindingsoluble ActRIIa polypeptides, activin-binding soluble ActRIIbpolypeptides, antibodies that bind to activin (particularly the activinA or B subunits, also referred to as PA or pB) and disrupt ActRIIaand/or ActRIb binding, antibodies that bind to ActRIIa and disruptactivin binding, antibodies that bind to ActRIIb and disrupt activinbinding, non-antibody proteins selected for activin, ActRIb or ActRIIabinding (see e.g., WO/2002/088171, WO/2006/055689, and WO/2002/032925for examples of such proteins and methods for design and selection ofsame), randomized peptides selected for activin, ActRIIb, or ActRIIabinding, often affixed to an Fc domain. Two different proteins (or othermoieties) with activin, ActRIb, or ActRIIa binding activity, especiallyactivin binders that block the type I (e.g., a soluble type I activinreceptor) and type II (e.g., a soluble type II activin receptor) bindingsites, respectively, may be linked together to create a bifunctionalbinding molecule. Nucleic acid aptamers, small molecules and otheragents that inhibit the activin-ActRII signaling axis are included asactivin-ActRII antagonists. Various proteins have activin-ActRIIantagonist activity, including inhibin (i.e., inhibin alpha subunit),although inhibin does not universally antagonize activin in all tissues,follistatin (e.g., follistatin-288 and follistatin-315), FSRP, activinC, alpha(2)-macroglobulin, and an M108A (methionine to alanine change atposition 108) mutant activin A. Generally, alternative forms of activin,particularly those with alterations in the type I receptor bindingdomain can bind to type II receptors and fail to form an active ternarycomplex, thus acting as antagonists. Additionally, nucleic acids, suchas antisense molecules, siRNAs or ribozymes that inhibit activin A, B, Cor E, or, particularly, ActRIIa or ActRIIb expression, can be used asactivin-ActRII antagonists. The activin-ActRII antagonist to be used mayexhibit selectivity for inhibiting activin-mediated signaling versusother members of the TGF-beta family, and particularly with respect toGDF8 and GDF11.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

2. ActRII Polypeptides

In certain aspects, the present invention relates to ActRIIpolypeptides. As used herein, the term “ActRII” refers to the family oftype II activin receptors. This family includes both the activinreceptor type IIa and the activin receptor type IIb.

In certain aspects, the present invention relates to ActRIIapolypeptides. As used herein, the term “ActRIIa” refers to a family ofactivin receptor type Ha (ActRIIa) proteins from any species andvariants derived from such ActRIIa proteins by mutagenesis or othermodification. Reference to ActRIIa herein is understood to be areference to any one of the currently identified forms. Members of theActRIIa family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ActRIIa polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ActRIIa family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. See, for example,WO/2006/012627. For example, ActRIIa polypeptides include polypeptidesderived from the sequence of any known ActRIIa having a sequence atleast about 80% identical to the sequence of an ActRIIa polypeptide, andoptionally at least 85%, 90%, 95%, 97%, 99% or greater identity. Forexample, an ActRIIa polypeptide of the invention may bind to and inhibitthe function of an ActRIIa protein and/or activin. An ActRIIapolypeptide may be selected for activity in promoting red blood cellformation in vivo. Examples of ActRIIa polypeptides include humanActRIIa precursor polypeptide (SEQ ID NO: 1) and soluble human ActRIIapolypeptides (e.g., SEQ ID NOs: 2, 3, 7 and 12).

The human ActRIIa precursor protein sequence is as follows:

(SEQ ID NO: 1) MGAAAKLAFAVFLISCSSGA ILGRSETQECLFFNANWEKDRT N QTGVEPCYGDKDKRRHCFATWK N ISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPYYNILLYSLVPLMLIAGIVICAFWVYRHHKMAYPPVLVPTQDPGPPPPSPLLGLKPLQLLEVKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRGTSVDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGLAYLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACIADFGLALKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQEVVVHKKKRPVLRDYWQKHAGMAMLCETIEECWDHDAEARLSAGCVGERITQMQRLTNIITTEDIVTVVTM VTNVDFPPKESSL

The signal peptide is single underlined; the extracellular domain is inbold and the potential N-linked glycosylation sites are doubleunderlined.

The human ActRIIa soluble (extracellular), processed polypeptidesequence is as follows:

(SEQ ID NO: 2) ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM EVTQPTSNPVTPKPP

The C-terminal “tail” of the extracellular domain is underlined. Thesequence with the “tail” deleted (a Δ15 sequence) is as follows:

(SEQ ID NO: 3) ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM

The nucleic acid sequence encoding human ActRIIa precursor protein is asfollows (nucleotides 164-1705 of Genbank entry NM_001616):

(SEQ ID NO: 4) ATGGGAGCTGCTGCAAAGTTGGCGTTTGCCGTCTTTCTTATCTCCTGTTCTTCAGGTGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCTATTACAACATCCTGCTCTATTCCTTGGTGCCACTTATGTTAATTGCGGGGATTGTCATTTGTGCATTTTGGGTGTACAGGCATCACAAGATGGCCTACCCTCCTGTACTTGTTCCAACTCAAGACCCAGGACCACCCCCACCTTCTCCATTACTAGGGTTGAAACCACTGCAGTTATTAGAAGTGAAAGCAAGGGGAAGATTTGGTTGTGTCTGGAAAGCCCAGTTGCTTAACGAATATGTGGCTGTCAAAATATTTCCAATACAGGACAAACAGTCATGGCAAAATGAATACGAAGTCTACAGTTTGCCTGGAATGAAGCATGAGAACATATTACAGTTCATTGGTGCAGAAAAACGAGGCACCAGTGTTGATGTGGATCTTTGGCTGATCACAGCATTTCATGAAAAGGGTTCACTATCAGACTTTCTTAAGGCTAATGTGGTCTCTTGGAATGAACTGTGTCATATTGCAGAAACCATGGCTAGAGGATTGGCATATTTACATGAGGATATACCTGGCCTAAAAGATGGCCACAAACCTGCCATATCTCACAGGGACATCAAAAGTAAAAATGTGCTGTTGAAAAACAACCTGACAGCTTGCATTGCTGACTTTGGGTTGGCCTTAAAATTTGAGGCTGGCAAGTCTGCAGGCGATACCCATGGACAGGTTGGTACCCGGAGGTACATGGCTCCAGAGGTATTAGAGGGTGCTATAAACTTCCAAAGGGATGCATTTTTGAGGATAGATATGTATGCCATGGGATTAGTCCTATGGGAACTGGCTTCTCGCTGTACTGCTGCAGATGGACCTGTAGATGAATACATGTTGCCATTTGAGGAGGAAATTGGCCAGCATCCATCTCTTGAAGACATGCAGGAAGTTGTTGTGCATAAAAAAAAGAGGCCTGTTTTAAGAGATTATTGGCAGAAACATGCTGGAATGGCAATGCTCTGTGAAACCATTGAAGAATGTTGGGATCACGACGCAGAAGCCAGGTTATCAGCTGGATGTGTAGGTGAAAGAATTACCCAGATGCAGAGACTAACAAATATTATTACCACAGAGGACATTGTAACAGTGGTCACAATGGTGACAAATGTTGACTTTCCTCCCAAAGAATCTAGTCTATGA

The nucleic acid sequence encoding a human ActRIIa soluble(extracellular) polypeptide is as follows:

(SEQ ID NO: 5) ATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCC

In certain aspects, the present invention relates to ActRIbpolypeptides. As used herein, the term “ActRIIb” refers to a family ofactivin receptor type IIb (ActRIb) proteins from any species andvariants derived from such ActRIb proteins by mutagenesis or othermodification. Reference to ActRIIb herein is understood to be areference to any one of the currently identified forms. Members of theActRIIb family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ActRIb polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ActRIIb family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. See, for example,WO/2006/012627. For example, ActRIb polypeptides include polypeptidesderived from the sequence of any known ActRIIb having a sequence atleast about 80% identical to the sequence of an ActRIIb polypeptide, andoptionally at least 85%, 90%, 95%, 97%, 99% or greater identity. Forexample, an ActRIIb polypeptide of the invention may bind to and inhibitthe function of an ActRIIb protein and/or activin. An ActRIIbpolypeptide may be selected for activity in promoting red blood cellformation in vivo. Examples of ActRIIb polypeptides include humanActRIIb precursor polypeptide (SEQ ID NO: 15) and soluble human ActRIIbpolypeptides (e.g., SEQ ID NO: 16, 17, 20, and 21).

The human ActRIIb precursor protein sequence is as follows:

(SEQ ID NO: 15)

ENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPPPSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMKHENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSYLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQHPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEERVSLIRRSVNGTTSDCLVSLVTSVTNVDLPPKESSI

The signal peptide is single underlined; the extracellular domain is inbold and the potential N-linked glycosylation sites are in boxes.

The human ActRIIb soluble (extracellular), processed polypeptidesequence is as follows:

(SEQ ID NO: 16) SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPE AGGPEVTYEPPPTAPT

The C-terminal “tail” of the extracellular domain is underlined. Thesequence with the “tail” deleted (a Δ15 sequence) is as follows:

(SEQ ID NO: 17) SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPE A

The nucleic acid sequence encoding a human ActRIIb precursor protein isas follows: (nucleotides 5-1543 of Genbank entry NM_001106)

(SEQ ID NO: 18) ATGACGGCGCCCTGGGTGGCCCTCGCCCTCCTCTGGGGATCGCTGTGGCCCGGCTCTGGGCGTGGGGAGGCTGAGACACGGGAGTGCATCTACTACAACGCCAACTGGGAGCTGGAGCGCACCAACCAGAGCGGCCTGGAGCGCTGCGAAGGCGAGCAGGACAAGCGGCTGCACTGCTACGCCTCCTGGGCCAACAGCTCTGGCACCATCGAGCTCGTGAAGAAGGGCTGCTGGCTAGATGACTTCAACTGCTACGATAGGCAGGAGTGTGTGGCCACTGAGGAGAACCCCCAGGTGTACTTCTGCTGCTGTGAAGGCAACTTCTGCAACGAGCGCTTCACTCATTTGCCAGAGGCTGGGGGCCCGGAAGTCACGTACGAGCCACCCCCGACAGCCCCCACCCTGCTCACGGTGCTGGCCTACTCACTGCTGCCCATCGGGGGCCTTTCCCTCATCGTCCTGCTGGCCTTTTGGATGTACCGGCATCGCAAGCCCCCCTACGGTCATGTGGACATCCATGAGGACCCTGGGCCTCCACCACCATCCCCTCTGGTGGGCCTGAAGCCACTGCAGCTGCTGGAGATCAAGGCTCGGGGGCGCTTTGGCTGTGTCTGGAAGGCCCAGCTCATGAATGACTTTGTAGCTGTCAAGATCTTCCCACTCCAGGACAAGCAGTCGTGGCAGAGTGAACGGGAGATCTTCAGCACACCTGGCATGAAGCACGAGAACCTGCTACAGTTCATTGCTGCCGAGAAGCGAGGCTCCAACCTCGAAGTAGAGCTGTGGCTCATCACGGCCTTCCATGACAAGGGCTCCCTCACGGATTACCTCAAGGGGAACATCATCACATGGAACGAACTGTGTCATGTAGCAGAGACGATGTCACGAGGCCTCTCATACCTGCATGAGGATGTGCCCTGGTGCCGTGGCGAGGGCCACAAGCCGTCTATTGCCCACAGGGACTTTAAAAGTAAGAATGTATTGCTGAAGAGCGACCTCACAGCCGTGCTGGCTGACTTTGGCTTGGCTGTTCGATTTGAGCCAGGGAAACCTCCAGGGGACACCCACGGACAGGTAGGCACGAGACGGTACATGGCTCCTGAGGTGCTCGAGGGAGCCATCAACTTCCAGAGAGATGCCTTCCTGCGCATTGACATGTATGCCATGGGGTTGGTGCTGTGGGAGCTTGTGTCTCGCTGCAAGGCTGCAGACGGACCCGTGGATGAGTACATGCTGCCCTTTGAGGAAGAGATTGGCCAGCACCCTTCGTTGGAGGAGCTGCAGGAGGTGGTGGTGCACAAGAAGATGAGGCCCACCATTAAAGATCACTGGTTGAAACACCCGGGCCTGGCCCAGCTTTGTGTGACCATCGAGGAGTGCTGGGACCATGATGCAGAGGCTCGCTTGTCCGCGGGCTGTGTGGAGGAGCGGGTGTCCCTGATTCGGAGGTCGGTCAACGGCACTACCTCGGACTGTCTCGTTTCCCTGGTGACCTCTGTCACCAATGTGGACCTGCCCCCTAAAGAGTCAAGCATCTAAThe nucleic acid sequence encoding a human ActRIIa soluble(extracellular) polypeptide is as follows:

(SEQ ID NO: 19) TCTGGGCGTGGGGAGGCTGAGACACGGGAGTGCATCTACTACAACGCCAACTGGGAGCTGGAGCGCACCAACCAGAGCGGCCTGGAGCGCTGCGAAGGCGAGCAGGACAAGCGGCTGCACTGCTACGCCTCCTGGGCCAACAGCTCTGGCACCATCGAGCTCGTGAAGAAGGGCTGCTGGCTAGATGACTTCAACTGCTACGATAGGCAGGAGTGTGTGGCCACTGAGGAGAACCCCCAGGTGTACTTCTGCTGCTGTGAAGGCAACTTCTGCAACGAGCGCTTCACTCATTTGCCAGAGGCTGGGGGCCCGGAAGTCACGTACGAGCCACCCCCGACAGCCCCCACC

In a specific embodiment, the invention relates to soluble ActRIIpolypeptides. As described herein, the term “soluble ActRII polypeptide”generally refers to polypeptides comprising an extracellular domain ofan ActRIIa or ActRIIb protein. The term “soluble ActRII polypeptide,” asused herein, includes any naturally occurring extracellular domain of anActRIIa or ActRIIb protein as well as any variants thereof (includingmutants, fragments and peptidomimetic forms). An activin-binding ActRIIpolypeptide is one that retains the ability to bind to activin,including, for example, activin AA, AB, BB, or forms that include a C orE subunit. Optionally, an activin-binding ActRII polypeptide will bindto activin AA with a dissociation constant of 1 nM or less. Theextracellular domain of an ActRII protein binds to activin and isgenerally soluble, and thus can be termed a soluble, activin-bindingActRII polypeptide. Examples of soluble, activin-binding ActRIIapolypeptides include the soluble polypeptides illustrated in SEQ ID NOs:2, 3, 7, 12 and 13. SEQ ID NO:7 is referred to as ActRIIa-hFc, and isdescribed further in the Examples. Other examples of soluble,activin-binding ActRIIa polypeptides comprise a signal sequence inaddition to the extracellular domain of an ActRIIa protein, for example,the honey bee mellitin leader sequence (SEQ ID NO: 8), the tissueplaminogen activator (TPA) leader (SEQ ID NO: 9) or the native ActRIIaleader (SEQ ID NO: 10). The ActRIIa-hFc polypeptide illustrated in SEQID NO:13 uses a TPA leader. Examples of soluble, activin-binding ActRIIbpolypeptides include the soluble polypeptides illustrated in SEQ ID NOs:16, 17, 20, and 21.

Functionally active fragments of ActRII polypeptides can be obtained byscreening polypeptides recombinantly produced from the correspondingfragment of the nucleic acid encoding an ActRII polypeptide. Inaddition, fragments can be chemically synthesized using techniques knownin the art such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. The fragments can be produced (recombinantly or by chemicalsynthesis) and tested to identify those peptidyl fragments that canfunction as antagonists (inhibitors) of ActRII protein or signalingmediated by activin.

Functionally active variants of ActRII polypeptides can be obtained byscreening libraries of modified polypeptides recombinantly produced fromthe corresponding mutagenized nucleic acids encoding an ActRIIpolypeptide. The variants can be produced and tested to identify thosethat can function as antagonists (inhibitors) of ActRII protein orsignaling mediated by activin. In certain embodiments, a functionalvariant of the ActRIIa polypeptides comprises an amino acid sequencethat is at least 75% identical to an amino acid sequence selected fromSEQ ID NOs: 2 or 3. In certain cases, the functional variant has anamino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from SEQ ID NOs: 2 or 3. Incertain embodiments, a functional variant of the ActRIIb polypeptidescomprises an amino acid sequence that is at least 75% identical to anamino acid sequence selected from SEQ ID NOs: 16 or 17. In certaincases, the functional variant has an amino acid sequence at least 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequenceselected from SEQ ID NOs: 17 or 18.

Functional variants may be generated by modifying the structure of anActRII polypeptide for such purposes as enhancing therapeutic efficacy,or stability (e.g., ex vivo shelf life and resistance to proteolyticdegradation in vivo). Such modified ActRII polypeptides when selected toretain activin binding, are considered functional equivalents of thenaturally-occurring ActRII polypeptides. Modified ActRII polypeptidescan also be produced, for instance, by amino acid substitution,deletion, or addition. For instance, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid(e.g., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains. Whether a change in the amino acidsequence of an ActRII polypeptide results in a functional homolog can bereadily determined by assessing the ability of the variant ActRIIpolypeptide to produce a response in cells in a fashion similar to thewild-type ActRII polypeptide.

In certain embodiments, the present invention contemplates specificmutations of the ActRII polypeptides so as to alter the glycosylation ofthe polypeptide. Such mutations may be selected so as to introduce oreliminate one or more glycosylation sites, such as O-linked or N-linkedglycosylation sites. Asparagine-linked glycosylation recognition sitesgenerally comprise a tripeptide sequence, asparagine-X-threonine orasparagine-X-serine (where “X” is any amino acid) which is specificallyrecognized by appropriate cellular glycosylation enzymes. The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the wild-type ActRIIpolypeptide (for O-linked glycosylation sites). A variety of amino acidsubstitutions or deletions at one or both of the first or third aminoacid positions of a glycosylation recognition site (and/or amino aciddeletion at the second position) results in non-glycosylation at themodified tripeptide sequence. Another means of increasing the number ofcarbohydrate moieties on an ActRII polypeptide is by chemical orenzymatic coupling of glycosides to the ActRII polypeptide. Depending onthe coupling mode used, the sugar(s) may be attached to (a) arginine andhistidine; (b) free carboxyl groups; (c) free sulfhydryl groups such asthose of cysteine; (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (f) the amide group ofglutamine. Removal of one or more carbohydrate moieties present on anActRII polypeptide may be accomplished chemically and/or enzymatically.Chemical deglycosylation may involve, for example, exposure of theActRII polypeptide to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the amino acid sequence intact.Enzymatic cleavage of carbohydrate moieties on ActRII polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al. (1987) Meth. Enzymol. 138:350. Thesequence of an ActRII polypeptide may be adjusted, as appropriate,depending on the type of expression system used, as mammalian, yeast,insect and plant cells may all introduce differing glycosylationpatterns that can be affected by the amino acid sequence of the peptide.In general, ActRII proteins for use in humans may be expressed in amammalian cell line that provides proper glycosylation, such as HEK293or CHO cell lines, although other mammalian expression cell lines areexpected to be useful as well.

This disclosure further contemplates a method of generating mutants,particularly sets of combinatorial mutants of an ActRII polypeptide, aswell as truncation mutants; pools of combinatorial mutants areespecially useful for identifying functional variant sequences. Thepurpose of screening such combinatorial libraries may be to generate,for example, ActRII polypeptide variants which bind to activin or otherligands. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, an ActRIIpolypeptide variant may be screened for ability to bind to an ActRIIligand, to prevent binding of an ActRII ligand to an ActRII polypeptideor to interfere with signaling caused by an ActRII ligand.

The activity of an ActRII polypeptide or its variants may also be testedin a cell-based or in vivo assay. For example, the effect of an ActRIIpolypeptide variant on the expression of genes involved in hematopoiesismay be assessed. This may, as needed, be performed in the presence ofone or more recombinant ActRII ligand proteins (e.g., activin), andcells may be transfected so as to produce an ActRII polypeptide and/orvariants thereof, and optionally, an ActRII ligand. Likewise, an ActRIIpolypeptide may be administered to a mouse or other animal, and one ormore blood measurements, such as an RBC count, hemoglobin, orreticulocyte count may be assessed.

Combinatorially-derived variants can be generated which have a selectiveor generally increased potency relative to a naturally occurring ActRIIpolypeptide. Likewise, mutagenesis can give rise to variants which haveintracellular half-lives dramatically different than the corresponding awild-type ActRII polypeptide. For example, the altered protein can berendered either more stable or less stable to proteolytic degradation orother cellular processes which result in destruction of, or otherwiseinactivation of a native ActRII polypeptide. Such variants, and thegenes which encode them, can be utilized to alter ActRII polypeptidelevels by modulating the half-life of the ActRII polypeptides. Forinstance, a short half-life can give rise to more transient biologicaleffects and, when part of an inducible expression system, can allowtighter control of recombinant ActRII polypeptide levels within thecell. In an Fc fusion protein, mutations may be made in the linker (ifany) and/or the Fc portion to alter the half-life of the protein.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential ActRII polypeptide sequences. For instance, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential ActRIIpolypeptide nucleotide sequences are expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display).

There are many ways by which the library of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes can then be ligated into anappropriate vector for expression. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc.3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevierpp273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura etal., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res.11:477). Such techniques have been employed in the directed evolution ofother proteins (see, for example, Scott et al., (1990) Science249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin etal., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, ActRII polypeptide variants can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis and the like (Ruf et al., (1994)Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al.,(1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol.Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838;and Cunningham et al., (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al.,(1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science232:316); by saturation mutagenesis (Meyers et al., (1986) Science232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol1:11-19); or by random mutagenesis, including chemical mutagenesis, etc.(Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press,Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in MolBiol 7:32-34). Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of ActRII polypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of ActRII polypeptides. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Preferredassays include activin binding assays and activin-mediated cellsignaling assays.

In certain embodiments, the ActRII polypeptides of the invention mayfurther comprise post-translational modifications in addition to anythat are naturally present in the ActRII polypeptides. Suchmodifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, the modified ActRII polypeptides may containnon-amino acid elements, such as polyethylene glycols, lipids, poly- ormono-saccharide, and phosphates. Effects of such non-amino acid elementson the functionality of an ActRII polypeptide may be tested as describedherein for other ActRII polypeptide variants. When an ActRII polypeptideis produced in cells by cleaving a nascent form of the ActRIIpolypeptide, post-translational processing may also be important forcorrect folding and/or function of the protein. Different cells (such asCHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specific cellularmachinery and characteristic mechanisms for such post-translationalactivities and may be chosen to ensure the correct modification andprocessing of the ActRII polypeptides.

In certain aspects, functional variants or modified forms of the ActRIIpolypeptides include fusion proteins having at least a portion of theActRII polypeptides and one or more fusion domains. Well known examplesof such fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, an immunoglobulin heavy chain constant region (Fc), maltosebinding protein (MBP), or human serum albumin. A fusion domain may beselected so as to confer a desired property. For example, some fusiondomains are particularly useful for isolation of the fusion proteins byaffinity chromatography. For the purpose of affinity purification,relevant matrices for affinity chromatography, such as glutathione-,amylase-, and nickel- or cobalt-conjugated resins are used. Many of suchmatrices are available in “kit” form, such as the Pharmacia GSTpurification system and the QIAexpress' system (Qiagen) useful with(HIS6) fusion partners. As another example, a fusion domain may beselected so as to facilitate detection of the ActRII polypeptides.Examples of such detection domains include the various fluorescentproteins (e.g., GFP) as well as “epitope tags,” which are usually shortpeptide sequences for which a specific antibody is available. Well knownepitope tags for which specific monoclonal antibodies are readilyavailable include FLAG, influenza virus haemagglutinin (HA), and c-myctags. In some cases, the fusion domains have a protease cleavage site,such as for Factor Xa or Thrombin, which allows the relevant protease topartially digest the fusion proteins and thereby liberate therecombinant proteins therefrom. The liberated proteins can then beisolated from the fusion domain by subsequent chromatographicseparation. In certain preferred embodiments, an ActRII polypeptide isfused with a domain that stabilizes the ActRII polypeptide in vivo (a“stabilizer” domain). By “stabilizing” is meant anything that increasesserum half life, regardless of whether this is because of decreaseddestruction, decreased clearance by the kidney, or other pharmacokineticeffect. Fusions with the Fc portion of an immunoglobulin are known toconfer desirable pharmacokinetic properties on a wide range of proteins.Likewise, fusions to human serum albumin can confer desirableproperties. Other types of fusion domains that may be selected includemultimerizing (e.g., dimerizing, tetramerizing) domains and functionaldomains (that confer an additional biological function, such as furtherstimulation of muscle growth).

As a specific example, the present invention provides a fusion proteincomprising a soluble extracellular domain of ActRIIa fused to an Fcdomain (e.g., SEQ ID NO: 6).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*

As an additional specific example, the present invention provides afusion protein comprising a soluble extracellular domain of ActRIIbfused to an Fc domain (e.g., SEQ ID NO: 21).

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Optionally, the Fc domain has one or more mutations at residues such asAsp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domainhaving one or more of these mutations (e.g., Asp-265 mutation) hasreduced ability of binding to the Fcγ receptor relative to a wildtype Fcdomain. In other cases, the mutant Fc domain having one or more of thesemutations (e.g., Asn-434 mutation) has increased ability of binding tothe MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fcdomain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an ActRII polypeptide may be placedC-terminal to a heterologous domain, or, alternatively, a heterologousdomain may be placed C-terminal to an ActRII polypeptide. The ActRIIpolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

In certain embodiments, the ActRII polypeptides of the present inventioncontain one or more modifications that are capable of stabilizing theActRII polypeptides. For example, such modifications enhance the invitro half life of the ActRII polypeptides, enhance circulatory halflife of the ActRII polypeptides or reducing proteolytic degradation ofthe ActRII polypeptides. Such stabilizing modifications include, but arenot limited to, fusion proteins (including, for example, fusion proteinscomprising an ActRII polypeptide and a stabilizer domain), modificationsof a glycosylation site (including, for example, addition of aglycosylation site to an ActRII polypeptide), and modifications ofcarbohydrate moiety (including, for example, removal of carbohydratemoieties from an ActRII polypeptide). As used herein, the term“stabilizer domain” not only refers to a fusion domain (e.g., Fc) as inthe case of fusion proteins, but also includes nonproteinaceousmodifications such as a carbohydrate moiety, or nonproteinaceous moiety,such as polyethylene glycol.

In certain embodiments, the present invention makes available isolatedand/or purified forms of the ActRII polypeptides, which are isolatedfrom, or otherwise substantially free of, other proteins. ActRIIpolypeptides will generally be produced by expression from recombinantnucleic acids.

3. Nucleic Acids Encoding ActRII Polypeptides

In certain aspects, the invention provides isolated and/or recombinantnucleic acids encoding any of the ActRII polypeptides (e.g., solubleActRIIa polypeptides and soluble ActRIIb polypeptides), includingfragments, functional variants and fusion proteins disclosed herein. Forexample, SEQ ID NO: 4 encodes the naturally occurring human ActRIIaprecursor polypeptide, while SEQ ID NO: 5 encodes the processedextracellular domain of ActRIIa. For example, SEQ ID NO: 18 encodes thenaturally occurring human ActRIIb precursor polypeptide, while SEQ IDNO: 19 encodes the processed extracellular domain of ActRIIb. Thesubject nucleic acids may be single-stranded or double stranded. Suchnucleic acids may be DNA or RNA molecules. These nucleic acids may beused, for example, in methods for making ActRII polypeptides or asdirect therapeutic agents (e.g., in a gene therapy approach).

In certain aspects, the subject nucleic acids encoding ActRIIapolypeptides are further understood to include nucleic acids that arevariants of SEQ ID NO: 4 or 5. In certain aspects, the subject nucleicacids encoding ActRIIb polypeptides are further understood to includenucleic acids that are variants of SEQ ID NO: 18 or 19. Variantnucleotide sequences include sequences that differ by one or morenucleotide substitutions, additions or deletions, such as allelicvariants.

In certain embodiments, the invention provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to SEQ ID NOs: 4, 5, 18, or 19. One of ordinaryskill in the art will appreciate that nucleic acid sequencescomplementary to SEQ ID NOs: 4, 5, 18, or 19, and variants of SEQ IDNOs: 4, 5, 18, or 19 are also within the scope of this invention. Infurther embodiments, the nucleic acid sequences of the invention can beisolated, recombinant, and/or fused with a heterologous nucleotidesequence, or in a DNA library.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence designated in SEQ ID NOs: 4, 5, 18, or 19,complement sequence of SEQ ID NOs: 4, 5, 18, or 19, or fragmentsthereof. As discussed above, one of ordinary skill in the art willunderstand readily that appropriate stringency conditions which promoteDNA hybridization can be varied. One of ordinary skill in the art willunderstand readily that appropriate stringency conditions which promoteDNA hybridization can be varied. For example, one could perform thehybridization at 6.0×sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the invention providesnucleic acids which hybridize under low stringency conditions of 6×SSCat room temperature followed by a wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 4, 5, 18, or 19 due to degeneracy in the genetic code arealso within the scope of the invention. For example, a number of aminoacids are designated by more than one triplet. Codons that specify thesame amino acid, or synonyms (for example, CAU and CAC are synonyms forhistidine) may result in “silent” mutations which do not affect theamino acid sequence of the protein. However, it is expected that DNAsequence polymorphisms that do lead to changes in the amino acidsequences of the subject proteins will exist among mammalian cells. Oneskilled in the art will appreciate that these variations in one or morenucleotides (up to about 3-5% of the nucleotides) of the nucleic acidsencoding a particular protein may exist among individuals of a givenspecies due to natural allelic variation. Any and all such nucleotidevariations and resulting amino acid polymorphisms are within the scopeof this invention.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the invention, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding an ActRII polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the ActRII polypeptide. Accordingly,the term regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding an ActRII polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid of the invention can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant ActRII polypeptide include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 3rdEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 2001). In some instances, it may be desirable toexpress the recombinant polypeptides by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors(such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ActRII polypeptides in CHO cells, such as a Pcmv-Scriptvector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen,Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As willbe apparent, the subject gene constructs can be used to cause expressionof the subject ActRII polypeptides in cells propagated in culture, e.g.,to produce proteins, including fusion proteins or variant proteins, forpurification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 4, 5, 18,or 19) for one or more of the subject ActRII polypeptides. The host cellmay be any prokaryotic or eukaryotic cell. For example, an ActRIIpolypeptide of the invention may be expressed in bacterial cells such asE. coli, insect cells (e.g., using a baculovirus expression system),yeast, or mammalian cells. Other suitable host cells are known to thoseskilled in the art.

Accordingly, the present invention further pertains to methods ofproducing the subject ActRII polypeptides. For example, a host celltransfected with an expression vector encoding an ActRIIa or ActRIIbpolypeptide can be cultured under appropriate conditions to allowexpression of the ActRII polypeptide to occur. The ActRII polypeptidemay be secreted and isolated from a mixture of cells and mediumcontaining the ActRII polypeptide. Alternatively, the ActRII polypeptidemay be retained cytoplasmically or in a membrane fraction and the cellsharvested, lysed and the protein isolated. A cell culture includes hostcells, media and other byproducts. Suitable media for cell culture arewell known in the art. The subject ActRII polypeptides can be isolatedfrom cell culture medium, host cells, or both, using techniques known inthe art for purifying proteins, including ion-exchange chromatography,gel filtration chromatography, ultrafiltration, electrophoresis,immunoaffinity purification with antibodies specific for particularepitopes of the ActRII polypeptides and affinity purification with anagent that binds to a domain fused to the ActRII polypeptide (e.g., aprotein A column may be used to purify an ActRIIa-Fc or ActRIIb-Fcfusion). In a preferred embodiment, the ActRII polypeptide is a fusionprotein containing a domain which facilitates its purification. In apreferred embodiment, purification is achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange. Asdemonstrated herein, ActRIIa-hFc protein was purified to a purityof >98% as determined by size exclusion chromatography and >95% asdetermined by SDS PAGE. This level of purity was sufficient to achievedesirable results in mice, rats and non-human primates.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ActRIIpolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified ActRII polypeptide (e.g., seeHochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Alternative Activin and ActRII Antagonists

The data presented herein demonstrates that antagonists ofactivin-ActRII signaling can be used to increase red blood cell orhemoglobin levels. Although soluble ActRIIa and ActRIIb polypeptides,and particularly ActRIIa-Fc and ActRIIb-Fc, are preferred antagonists,and although such antagonists may affect red blood cell levels through amechanism other than activin antagonism (e.g., activin inhibition may bean indicator of the tendency of an agent to inhibit the activities of aspectrum of molecules, including, perhaps, other members of the TGF-betasuperfamily, and such collective inhibition may lead to the desiredeffect on hematopoiesis), other types of activin-ActRII antagonists areexpected to be useful, including anti-activin (e.g., activin PA, OB, f cand PE) antibodies, anti-ActRIIa antibodies, anti-ActRIIb antibodies,antisense, RNAi or ribozyme nucleic acids that inhibit the production ofActRIIa and/or ActRIb, and other inhibitors of activin, ActRIIb orActRIIa, particularly those that disrupt activin-ActRIIa and/oractivin-ActRIIb binding.

An antibody that is specifically reactive with an ActRII polypeptide(e.g., a soluble ActRIIa or ActRIIb polypeptide) and which either bindscompetitively to ligand with the ActRII polypeptide or otherwiseinhibits ActRII-mediated signaling may be used as an antagonist ofActRII polypeptide activities. Likewise, an antibody that isspecifically reactive with an activin β_(A), β_(B), β_(C) or β_(E)polypeptide, or any heterodimer thereof, and which disrupts ActRIIaand/or ActRIIb binding may be used as an antagonist.

By using immunogens derived from an ActRIIa polypeptide, ActRIIbpolypeptide or an activin polypeptide, anti-protein/anti-peptideantisera or monoclonal antibodies can be made by standard protocols(see, for example, Antibodies: A Laboratory Manual ed. by Harlow andLane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, ahamster or rabbit can be immunized with an immunogenic form of theactivin, ActRIIa or ActRIIb polypeptide, an antigenic fragment which iscapable of eliciting an antibody response, or a fusion protein.Techniques for conferring immunogenicity on a protein or peptide includeconjugation to carriers or other techniques well known in the art. Animmunogenic portion of an ActRII or activin polypeptide can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassays can be used with the immunogen asantigen to assess the levels of antibodies.

Following immunization of an animal with an antigenic preparation of anactivin, ActRIIa or ActRIIb polypeptide, antisera can be obtained and,if desired, polyclonal antibodies can be isolated from the serum. Toproduce monoclonal antibodies, antibody-producing cells (lymphocytes)can be harvested from an immunized animal and fused by standard somaticcell fusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with an activin, ActRIIaor ActRIIb polypeptide and monoclonal antibodies isolated from a culturecomprising such hybridoma cells.

The term “antibody” as used herein is intended to include wholeantibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includesfragments or domains of immunoglobulins which are reactive with aselected antigen. Antibodies can be fragmented using conventionaltechniques and the fragments screened for utility and/or interactionwith a specific epitope of interest. Thus, the term includes segments ofproteolytically-cleaved or recombinantly-prepared portions of anantibody molecule that are capable of selectively reacting with acertain protein. Non-limiting examples of such proteolytic and/orrecombinant fragments include Fab, F(ab′)2, Fab′, Fv, and single chainantibodies (scFv) containing a V[L] and/or V[H] domain joined by apeptide linker. The scFv's may be covalently or non-covalently linked toform antibodies having two or more binding sites. The term antibody alsoincludes polyclonal, monoclonal, or other purified preparations ofantibodies and recombinant antibodies. The term “recombinant antibody”,means an antibody, or antigen binding domain of an immunoglobulin,expressed from a nucleic acid that has been constructed using thetechniques of molecular biology, such as a humanized antibody or a fullyhuman antibody developed from a single chain antibody. Single domain andsingle chain antibodies are also included within the term “recombinantantibody”.

In certain embodiments, an antibody of the invention is a monoclonalantibody, and in certain embodiments, the invention makes availablemethods for generating novel antibodies. For example, a method forgenerating a monoclonal antibody that binds specifically to an ActRIIapolypeptide, ActRIIb polypeptide, or activin polypeptide may compriseadministering to a mouse an amount of an immunogenic compositioncomprising the antigen polypeptide effective to stimulate a detectableimmune response, obtaining antibody-producing cells (e.g., cells fromthe spleen) from the mouse and fusing the antibody-producing cells withmyeloma cells to obtain antibody-producing hybridomas, and testing theantibody-producing hybridomas to identify a hybridoma that produces amonocolonal antibody that binds specifically to the antigen. Onceobtained, a hybridoma can be propagated in a cell culture, optionally inculture conditions where the hybridoma-derived cells produce themonoclonal antibody that binds specifically to the antigen. Themonoclonal antibody may be purified from the cell culture.

The adjective “specifically reactive with” as used in reference to anantibody is intended to mean, as is generally understood in the art,that the antibody is sufficiently selective between the antigen ofinterest (e.g., an activin, ActRIIa or ActRIIb polypeptide) and otherantigens that are not of interest that the antibody is useful for, atminimum, detecting the presence of the antigen of interest in aparticular type of biological sample. In certain methods employing theantibody, such as therapeutic applications, a higher degree ofspecificity in binding may be desirable. Monoclonal antibodies generallyhave a greater tendency (as compared to polyclonal antibodies) todiscriminate effectively between the desired antigens and cross-reactingpolypeptides. One characteristic that influences the specificity of anantibody:antigen interaction is the affinity of the antibody for theantigen. Although the desired specificity may be reached with a range ofdifferent affinities, generally preferred antibodies will have anaffinity (a dissociation constant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ M orless.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore™ binding assay, Biacore AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

Examples of categories of nucleic acid compounds that are activin orActRII antagonists include antisense nucleic acids, RNAi constructs andcatalytic nucleic acid constructs. A nucleic acid compound may be singleor double stranded. A double stranded compound may also include regionsof overhang or non-complementarity, where one or the other of thestrands is single stranded. A single stranded compound may includeregions of self-complementarity, meaning that the compound forms aso-called “hairpin” or “stem-loop” structure, with a region of doublehelical structure. A nucleic acid compound may comprise a nucleotidesequence that is complementary to a region consisting of no more than1000, no more than 500, no more than 250, no more than 100, or no morethan 50, 35, 25, 22, 20, 18 or 15 nucleotides of the full-length ActRIInucleic acid sequence or activin β_(A), β_(B), β_(C), or β_(E) nucleicacid sequence. The region of complementarity will preferably be at least8 nucleotides, and optionally about 18 to 35 nucleotides. A region ofcomplementarity may fall within an intron, a coding sequence or anoncoding sequence of the target transcript, such as the coding sequenceportion. Generally, a nucleic acid compound will have a length of about8 to about 500 nucleotides or base pairs in length, and optionally thelength will be about 14 to about 50 nucleotides. A nucleic acid may be aDNA (particularly for use as an antisense), RNA or RNA:DNA hybrid. Anyone strand may include a mixture of DNA and RNA, as well as modifiedforms that cannot readily be classified as either DNA or RNA. Likewise,a double stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA, and anyone strand may also include a mixture of DNA and RNA, as well asmodified forms that cannot readily be classified as either DNA or RNA. Anucleic acid compound may include any of a variety of modifications,including one or modifications to the backbone (the sugar-phosphateportion in a natural nucleic acid, including internucleotide linkages)or the base portion (the purine or pyrimidine portion of a naturalnucleic acid). An antisense nucleic acid compound will preferably have alength of about 15 to about 30 nucleotides and will often contain one ormore modifications to improve characteristics such as stability in theserum, in a cell or in a place where the compound is likely to bedelivered, such as the stomach in the case of orally delivered compoundsand the lung for inhaled compounds. In the case of an RNAi construct,the strand complementary to the target transcript will generally be RNAor modifications thereof. The other strand may be RNA, DNA or any othervariation. The duplex portion of double stranded or single stranded“hairpin” RNAi construct will generally have a length of 18 to 40nucleotides in length and optionally about 21 to 23 nucleotides inlength, so long as it serves as a Dicer substrate. Catalytic orenzymatic nucleic acids may be ribozymes or DNA enzymes and may alsocontain modified forms. Nucleic acid compounds may inhibit expression ofthe target by about 50%, 75%, 90% or more when contacted with cellsunder physiological conditions and at a concentration where a nonsenseor sense control has little or no effect. Preferred concentrations fortesting the effect of nucleic acid compounds are 1, 5 and 10 micromolar.Nucleic acid compounds may also be tested for effects on, for example,red blood cell levels.

5. Screening Assays

In certain aspects, the present invention relates to the use of ActRIIpolypeptides (e.g., soluble ActRIIa or ActRIIb polypeptides) and activinpolypeptides to identify compounds (agents) which are agonist orantagonists of the activin-ActRIIa and/or activin ActRIb signalingpathway. Compounds identified through this screening can be tested toassess their ability to modulate red blood cell, hemoglobin and/orreticulocyte levels in vivo or in vitro. These compounds can be tested,for example, in animal models.

There are numerous approaches to screening for therapeutic agents forincreasing red blood cell or hemoglobin levels by targeting activin andActRII signaling. In certain embodiments, high-throughput screening ofcompounds can be carried out to identify agents that perturb activin orActRII-mediated effects on a selected cell line. In certain embodiments,the assay is carried out to screen and identify compounds thatspecifically inhibit or reduce binding of an ActRIIa or ActRIIbpolypeptide to activin. Alternatively, the assay can be used to identifycompounds that enhance binding of an ActRIIa or ActRIIb polypeptide toactivin. In a further embodiment, the compounds can be identified bytheir ability to interact with an activin, ActRIIb polypeptide, orActRIIa polypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. In aspecific embodiment, the test agent is a small organic molecule having amolecular weight of less than about 2,000 Daltons.

The test compounds of the invention can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase (GST),photoactivatible crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between an ActRIIapolypeptide and activin and/or between an ActRIb polypeptide andactivin.

Merely to illustrate, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an isolated andpurified ActRIIa polypeptide which is ordinarily capable of binding toactivin. To the mixture of the compound and ActRIIa polypeptide is thenadded a composition containing an ActRIIa ligand. Detection andquantification of ActRIIa/activin complexes provides a means fordetermining the compound's efficacy at inhibiting (or potentiating)complex formation between the ActRIIa polypeptide and activin. Theefficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide abaseline for comparison. For example, in a control assay, isolated andpurified activin is added to a composition containing the ActRIIapolypeptide, and the formation of ActRIIa/activin complex is quantitatedin the absence of the test compound. It will be understood that, ingeneral, the order in which the reactants may be admixed can be varied,and can be admixed simultaneously. Moreover, in place of purifiedproteins, cellular extracts and lysates may be used to render a suitablecell-free assay system. Compounds that affect ActRIb signaling may beidentified in a similar manner using an ActRIb polypeptide and an ActRIbligand.

Complex formation between the ActRII polypeptide and activin may bedetected by a variety of techniques. For instance, modulation of theformation of complexes can be quantitated using, for example, detectablylabeled proteins such as radiolabeled (e.g., ³²P, ³⁵S, ⁴¹C or ³H),fluorescently labeled (e.g., FITC), or enzymatically labeled ActRIIa orActRIIb polypeptide or activin, by immunoassay, or by chromatographicdetection.

In certain embodiments, the present invention contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between an ActRII polypeptide and its bindingprotein. Further, other modes of detection, such as those based onoptical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.5,677,196), surface plasmon resonance (SPR), surface charge sensors, andsurface force sensors, are compatible with many embodiments of theinvention.

Moreover, the present invention contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between an ActRII polypeptide andits binding protein. See for example, U.S. Pat. No. 5,283,317; Zervos etal. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; andIwabuchi et al. (1993) Oncogene 8:1693-1696). In a specific embodiment,the present invention contemplates the use of reverse two hybrid systemsto identify compounds (e.g., small molecules or peptides) thatdissociate interactions between an ActRII polypeptide and its bindingprotein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; andU.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, the subject compounds are identified by theirability to interact with an ActRII or activin polypeptide of theinvention. The interaction between the compound and the ActRIIa,ActRIIb, or activin polypeptide may be covalent or non-covalent. Forexample, such interaction can be identified at the protein level usingin vitro biochemical methods, including photo-crosslinking, radiolabeledligand binding, and affinity chromatography (Jakoby W B et al., 1974,Methods in Enzymology 46: 1). In certain cases, the compounds may bescreened in a mechanism based assay, such as an assay to detectcompounds which bind to an activin or ActRII polypeptide. This mayinclude a solid phase or fluid phase binding event. Alternatively, thegene encoding an activin or ActRII polypeptide can be transfected with areporter system (e.g., β-galactosidase, luciferase, or green fluorescentprotein) into a cell and screened against the library optionally by ahigh throughput screening or with individual members of the library.Other mechanism based binding assays may be used, for example, bindingassays which detect changes in free energy. Binding assays can beperformed with the target fixed to a well, bead or chip or captured byan immobilized antibody or resolved by capillary electrophoresis. Thebound compounds may be detected usually using colorimetric orfluorescence or surface plasmon resonance.

6. Exemplary Therapeutic Uses

In certain embodiments, activin-ActRII antagonists (e.g., ActRIIa orActRIIb polypeptides) of the present invention can be used to increasered blood cell levels in mammals such as rodents and primates, andparticularly human patients. In certain embodiments, the presentinvention provides methods of treating or preventing anemia in anindividual in need thereof by administering to the individual atherapeutically effective amount of an activin-ActRIIa antagonist, suchas an ActRIIa polypeptide, or a therapeutically effective amount of anactivin-ActRIIb antagonist, such as an ActRIIb polypeptide. In certainembodiments, the present invention provides methods of promoting redblood cell formation in an individual by administering to the individuala therapeutically effective amount of an activin-ActRII antagonist,particularly an ActRII polypeptide. These methods may be used fortherapeutic and prophylactic treatments of mammals, and particularlyhumans.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes prophylaxis of the named condition or amelioration orelimination of the condition once it has been established. In eithercase, prevention or treatment may be discerned in the diagnosis providedby a physician or other health care provider and the intended result ofadministration of the therapeutic agent.

As shown herein, activin-ActRIIa antagonists and activin-ActRIIbantagonists may be used to increase red blood cell, hemoglobin orreticulocyte levels in healthy individuals, and such antagonists may beused in selected patient populations. Examples of appropriate patientpopulations include those with undesirably low red blood cell orhemoglobin levels, such as patients having an anemia, and those that areat risk for developing undesirably low red blood cell or hemoglobinlevels, such as those patients that are about to undergo major surgeryor other procedures that may result in substantial blood loss. In oneembodiment, a patient with adequate red blood cell levels is treatedwith an activin-ActRIIa antagonist to increase red blood cell levels,and then blood is drawn and stored for later use in transfusions. In oneembodiment, a patient with adequate red blood cell levels is treatedwith an activin-ActRIIb antagonist to increase red blood cell levels,and then blood is drawn and stored for later use in transfusions.

Activin-ActRII antagonists disclosed herein, and particularly ActRIIa-Fcand ActRIIb proteins, may be used to increase red blood cell levels inpatients having an anemia. When observing hemoglobin levels in humans, alevel of less than normal for the appropriate age and gender categorymay be indicative of anemia, although individual variations are takeninto account. For example, a hemoglobin level of 12 g/dl is generallyconsidered the lower limit of normal in the general adult population.Potential causes include blood-loss, nutritional deficits, medicationreaction, various problems with the bone marrow and many diseases. Moreparticularly, anemia has been associated with a variety of disordersthat include, for example, chronic renal failure, myelodysplasticsyndrome, rheumatoid arthritis, bone marrow transplantation. Anemia mayalso be associated with the following conditions: solid tumors (e.g.breast cancer, lung cancer, colon cancer); tumors of the lymphaticsystem (e.g. chronic lymphocyte leukemia, non-Hodgkins and Hodgkinslymphomas); tumors of the hematopoietic system (eg. leukemia,myelodysplastic syndrome, multiple myeloma); radiation therapy;chemotherapy (e.g. platinum containing regimens); inflammatory andautoimmune diseases, including, but not limited to, rheumatoidarthritis, other inflammatory arthritides, systemic lupus erythematosis(SLE), acute or chronic skin diseases (e.g. psoriasis), inflammatorybowel disease (e.g. Crohn's disease and ulcerative colitis); acute orchronic renal disease or failure including idiopathic or congenitalconditions; acute or chronic liver disease; acute or chronic bleeding;situations where transfusion of red blood cells is not possible due topatient allo- or auto-antibodies and/or for religious reasons (e.g. someJehovah's Witnesses); infections (e.g. malaria, osteomyelitis);hemoglobinopathies, including, for example, sickle cell disease,thalassemias; drug use or abuse, e.g. alcohol misuse; pediatric patientswith anemia from any cause to avoid transfusion; and elderly patients orpatients with underlying cardiopulmonary disease with anemia who cannotreceive transfusions due to concerns about circulatory overload.

Patients may be treated with a dosing regimen intended to restore thepatient to a target hemoglobin level, usually between about 10 g/dl andabout 12.5 g/dl, and typically about 11.0 g/dl (see also Jacobs et al.(2000) Nephrol Dial Transplant 15, 15-19), although lower target levelsmay cause fewer cardiovascular side effects. Alternatively, hematocritlevels (percentage of the volume of a blood sample occupied by thecells) can be used as a measure for the condition of red blood cells.Hematocrit levels for healthy individuals range from 41 to 51% for adultmales and from 35 to 45% for adult females. Target hematocrit levels areusually around 30-33%. Moreover, hemoglobin/hematocrit levels vary fromperson to person. Thus, optimally, the target hemoglobin/hematocritlevel can be individualized for each patient.

The rapid effect on red blood cell levels of the activin-ActRIIaantagonists disclosed herein indicate that these agents act by adifferent mechanism than Epo. Accordingly, these antagonists may beuseful for increasing red blood cell and hemoglobin levels in patientsthat do not respond well to Epo. For example, an activin-ActRIIaantagonist may be beneficial for a patient in which administering of anormal to increased (>300 IU/kg/week) dose of Epo does not result in theincrease of hemoglobin level up to the target level. Patients with aninadequate Epo response are found for all types of anemia, but highernumbers of non-responders have been observed particularly frequently inpatients with cancers and patients with end-stage renal disease. Aninadequate response to Epo can be either constitutive (i.e. observedupon the first treatment with Epo) or acquired (e.g. observed uponrepeated treatment with Epo).

The activin-ActRII antagonists may also be used to treat patients thatare susceptible to adverse effects of Epo. The primary adverse effectsof Epo are an excessive increase in the hematocrit or hemoglobin levelsand polycythemia. Elevated hematocrit levels can lead to hypertension(more particularly aggravation of hypertension) and vascular thrombosis.Other adverse effects of Epo which have been reported, some of whichrelated to hypertension, are headaches, influenza-like syndrome,obstruction of shunts, myocardial infarctions and cerebral convulsionsdue to thrombosis, hypertensive encephalopathy, and red cell blood cellapplasia (Singibarti, (1994) J. Clin Investig 72(suppl 6), S36-S43; Horlet al. (2000) Nephrol Dial Transplant 15(suppl 4), 51-56; Delanty et al.(1997) Neurology 49, 686-689; Bunn (2002) N Engl J Med 346(7), 522-523).

7. Pharmaceutical Compositions

In certain embodiments, activin-ActRII antagonists (e.g., ActRIIa andActRIIb polypeptides) of the present invention are formulated with apharmaceutically acceptable carrier. For example, an ActRII polypeptidecan be administered alone or as a component of a pharmaceuticalformulation (therapeutic composition). The subject compounds may beformulated for administration in any convenient way for use in human orveterinary medicine.

In certain embodiments, the therapeutic method of the invention includesadministering the composition systemically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Therapeutically useful agents other than the activin-ActRIIantagonists which may also optionally be included in the composition asdescribed above, may be administered simultaneously or sequentially withthe subject compounds (e.g., ActRIIa and ActRIIb polypeptides) in themethods of the invention.

Typically, activin-ActRII antagonists will be administered parenterally.Pharmaceutical compositions suitable for parenteral administration maycomprise one or more ActRII polypeptides in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

Further, the composition may be encapsulated or injected in a form fordelivery to a target tissue site (e.g., bone marrow). In certainembodiments, compositions of the present invention may include a matrixcapable of delivering one or more therapeutic compounds (e.g., ActRIIaor ActRIIb polypeptides) to a target tissue site (e.g., bone marrow),providing a structure for the developing tissue and optimally capable ofbeing resorbed into the body. For example, the matrix may provide slowrelease of the ActRII polypeptides. Such matrices may be formed ofmaterials presently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof, and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the invention (e.g., ActRIIa and ActRIbpolypeptides). The various factors include, but are not limited to, thepatient's red blood cell count, hemoglobin level or other diagnosticassessments, the desired target red blood cell count, the patient's age,sex, and diet, the severity of any disease that may be contributing to adepressed red blood cell level, time of administration, and otherclinical factors. The addition of other known growth factors to thefinal composition may also affect the dosage. Progress can be monitoredby periodic assessment of red blood cell and hemoglobin levels, as wellas assessments of reticulocyte levels and other indicators of thehematopoietic process.

Experiments with primates and humans have demonstrated that effects ofActRIIa-Fc on red blood cell levels are detectable when the compound isdosed at intervals and amounts sufficient to achieve serumconcentrations of about 100 ng/ml or greater, for a period of at leastabout 20 to 30 days. Dosing to obtain serum levels of 200 ng/ml, 500ng/ml, 1000 ng/ml or greater for a period of at least 20 to 30 days mayalso be used. Bone effects can be observed at serum levels of about 200ng/ml, with substantial effects beginning at about 1000 ng/ml or higher,over a period of at least about 20 to 30 days. Thus, if it is desirableto achieve effects on red blood cells while having little effect onbone, a dosing scheme may be designed to deliver a serum concentrationof between about 100 and 1000 ng/ml over a period of about 20 to 30days. In humans, serum levels of 200 ng/ml may be achieved with a singledose of 0.1 mg/kg or greater and serum levels of 1000 ng/ml may beachieved with a single dose of 0.3 mg/kg or greater. The observed serumhalf-life of the molecule is between about 20 and 30 days, substantiallylonger than most Fc fusion proteins, and thus a sustained effectiveserum level may be achieved, for example, by dosing with about 0.05 to0.5 mg/kg on a weekly or biweekly basis, or higher doses may be usedwith longer intervals between dosings. For example, doses of 0.1 to 1mg/kg might be used on a monthly or bimonthly basis.

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of ActRII polypeptides. Such therapy wouldachieve its therapeutic effect by introduction of the ActRIIa or ActRIIbpolynucleotide sequences into cells or tissues having the disorders aslisted above. Delivery of ActRII polynucleotide sequences can beachieved using a recombinant expression vector such as a chimeric virusor a colloidal dispersion system. Preferred for therapeutic delivery ofActRII polynucleotide sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or an RNA virus suchas a retrovirus. The retroviral vector may be a derivative of a murineor avian retrovirus. Examples of retroviral vectors in which a singleforeign gene can be inserted include, but are not limited to: Moloneymurine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). Anumber of additional retroviral vectors can incorporate multiple genes.All of these vectors can transfer or incorporate a gene for a selectablemarker so that transduced cells can be identified and generated.Retroviral vectors can be made target-specific by attaching, forexample, a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody. Those of skill in the art willrecognize that specific polynucleotide sequences can be inserted intothe retroviral genome or attached to a viral envelope to allow targetspecific delivery of the retroviral vector containing the ActRIIpolynucleotide.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for ActRII polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1: ActRIIa-Fc Fusion Proteins

Applicants constructed a soluble ActRIIa fusion protein that has theextracellular domain of human ActRIIa fused to a human or mouse Fcdomain with a minimal linker in between. The constructs are referred toas ActRIIa-hFc and ActRIIa-mFc, respectively.

ActRIIa-hFc is shown below as purified from CHO cell lines (SEQ ID NO:7):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The ActRIIa-hFc and ActRIIa-mFc proteins were expressed in CHO celllines. Three different leader sequences were considered:

(i) Honey bee mellitin (HBML): (SEQ ID NO: 8) MKFLVNVALVFMVVYISYIYA(ii) Tissue Plasminogen Activator (TPA): (SEQ ID NO: 9)MDAMKRGLCCVLLLCGAVFVSP (iii) Native: (SEQ ID NO: 10)MGAAAKLAFAVFLISCSSGA.

The selected form employs the TPA leader and has the followingunprocessed amino acid sequence:

(SEQ ID NO: 13) MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK

This polypeptide is encoded by the following nucleic acid sequence:

(SEQ ID NO: 14) ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGTCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGAGAATTC

Both ActRIIa-hFc and ActRIIa-mFc were remarkably amenable to recombinantexpression. As shown in FIG. 1A, the protein was purified as a single,well-defined peak of protein. N-terminal sequencing revealed a singlesequence of -ILGRSETQE (SEQ ID NO: 11). Purification could be achievedby a series of column chromatography steps, including, for example,three or more of the following, in any order: protein A chromatography,Q sepharose chromatography, phenylsepharose chromatography, sizeexclusion chromatography, and cation exchange chromatography. Thepurification could be completed with viral filtration and bufferexchange. The ActRIIa-hFc protein was purified to a purity of >98% asdetermined by size exclusion chromatography and >95% as determined bySDS PAGE.

ActRIIa-hFc and ActRIIa-mFc showed a high affinity for ligands,particularly activin A. GDF-11 or Activin A (“ActA”) were immobilized ona Biacore CM5 chip using standard amine coupling procedure. ActRIIa-hFcand ActRIIa-mFc proteins were loaded onto the system, and binding wasmeasured. ActRIIa-hFc bound to activin with a dissociation constant(K_(D)) of 5×10¹², and the protein bound to GDF11 with a K_(D) of9.96×10⁻⁹. See FIG. 2A and FIG. 2B. ActRIIa-mFc behaved similarly.

The ActRIIa-hFc was very stable in pharmacokinetic studies. Rats weredosed with 1 mg/kg, 3 mg/kg or 10 mg/kg of ActRIIa-hFc protein andplasma levels of the protein were measured at 24, 48, 72, 144 and 168hours. In a separate study, rats were dosed at 1 mg/kg, 10 mg/kg or 30mg/kg. In rats, ActRIIa-hFc had an 11-14 day serum half life andcirculating levels of the drug were quite high after two weeks (11μg/ml, 110 μg/ml or 304 μg/ml for initial administrations of 1 mg/kg, 10mg/kg or 30 mg/kg, respectively.) In cynomolgus monkeys, the plasma halflife was substantially greater than 14 days and circulating levels ofthe drug were 25 μg/ml, 304 μg/ml or 1440 μg/ml for initialadministrations of 1 mg/kg, 10 mg/kg or 30 mg/kg, respectively.

Example 2: ActRIIa-hFc Increases Red Blood Cell Levels in Non-HumanPrimates

The study employed four groups of five male and five female cynomolgusmonkeys each, with three per sex per group scheduled for termination onDay 29, and two per sex per group scheduled for termination on Day 57.Each animal was administered the vehicle (Group I) or ActRIIa-Fc atdoses of 1, 10, or 30 mg/kg (Groups 2, 3 and 4, respectively) viaintravenous (IV) injection on Days 1, 8, 15 and 22. The dose volume wasmaintained at 3 mL/kg. Various measures of red blood cell levels wereassessed two days prior to the first administration and at days 15, 29and 57 (for the remaining two animals) after the first administration.

The ActRIIa-hFc causes statistically significant increases in mean redblood cell parameters (red blood cell count [RBC], hemoglobin [HGB], andhematocrit [HCT]) for males and females, at all dose levels and timepoints throughout the study, with accompanying elevations in absoluteand relative reticulocyte counts (ARTC; RTC). See FIG. 3A, FIG. 3B, FIG.4A, FIG. 4B, FIG. 5A, FIG. 5B, FIG. 6A, and FIG. 6B.

Statistical significance was calculated for each treatment grouprelative to the mean for the treatment group at baseline.

Notably, the increases in red blood cell counts and hemoglobin levelsare roughly equivalent in magnitude to effects reported witherythropoietin. The onset of these effects is more rapid with ActRIIa-Fcthan with erythropoietin.

Similar results were observed with rats and mice.

Example 3: ActRIIa-hFc Increases Red Blood Cell Levels in Human Patients

The ActRIIa-hFc fusion protein described in Example 1 was administeredto human patients in a randomized, double-blind, placebo-controlledstudy that was conducted to evaluate, primarily, the safety of theprotein in healthy, postmenopausal women. Forty-eight subjects wererandomized in cohorts of 6 to receive either a single dose ofActRIIa-hFc or placebo (5 active:1 placebo). Dose levels ranged from0.01 to 3.0 mg/kg intravenously (IV) and 0.03 to 0.1 mg/kgsubcutaneously (SC). All subjects were followed for 120 days. Inaddition to pharmacokinetic (PK) analyses, the biologic activity ofActRIIa-hFc was also assessed by measurement of biochemical markers ofbone formation and resorption, and FSH levels.

To look for potential changes, hemoglobin and RBC numbers were examinedin detail for all subjects over the course of the study and compared tothe baseline levels. Platelet counts were compared over the same time asthe control. There were no clinically significant changes from thebaseline values over time for the platelet counts.

PK analysis of ActRIIa-hFc displayed a linear profile with dose, and amean half-life of approximately 25-32 days. The area-under-curve (AUC)for ActRIIa-hFc was linearly related to dose, and the absorption afterSC dosing was essentially complete (see FIGS. 7 and 8). These dataindicate that SC is a desirable approach to dosing because it providesequivalent bioavailability and serum-half life for the drug whileavoiding the spike in serum concentrations of drug associated with thefirst few days of IV dosing (see FIG. 8). ActRIIa-hFc caused a rapid,sustained dose-dependent increase in serum levels of bone-specificalkaline phosphatase (BAP), which is a marker for anabolic bone growth,and a dose-dependent decrease in C-terminal type 1 collagen telopeptideand tartrate-resistant acid phosphatase 5b levels, which are markers forbone resorption. Other markers, such as P1NP showed inconclusiveresults. BAP levels showed near saturating effects at the highest dosageof drug, indicating that half-maximal effects on this anabolic bonebiomarker could be achieved at a dosage of 0.3 mg/kg, with increasesranging up to 3 mg/kg. Calculated as a relationship of pharmacodynamiceffect to AUC for drug, the EC50 is 51,465 (day*ng/ml). See FIG. 9.These bone biomarker changes were sustained for approximately 120 daysat the highest dose levels tested. There was also a dose-dependentdecrease in serum FSH levels consistent with inhibition of activin.

Overall, there was a very small non-drug related reduction in hemoglobinover the first week of the study probably related to study phlebotomy inthe 0.01 and 0.03 mg/kg groups whether given IV or SC. The 0.1 mg/kg SCand IV hemoglobin results were stable or showed modest increases by Day8-15. At the 0.3 mg/kg IV dose level there was a clear increase in HGBlevels seen as early as Day 2 and often peaking at Day 15-29 that wasnot seen in the placebo subjects. At this point in the study, thischange has not reached statistical significance.

Overall, ActRIIa-hFc showed a dose-dependent effect on red blood cellcounts and reticulocyte counts. For a summary of hematological changes,see FIGS. 10-13.

Example 4: Alternative ActRIIa-Fc Proteins

A variety of ActRIIa variants that may be used according to the methodsdescribed herein are described in the International Patent Applicationpublished as WO2006/012627 (see e.g., pp. 55-58), incorporated herein byreference in its entirety. An alternative construct may have a deletionof the C-terminal tail (the final 15 amino acids of the extracellulardomain of ActRIIa. The sequence for such a construct is presented below(Fc portion underlined)(SEQ ID NO: 12):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Example 5: ActRIIb-Fc Fusion Proteins

Applicants constructed a soluble ActRIIb fusion protein that has theextracellular domain of human ActRIIb fused to a human Fc domain Aco-crystal structure of Activin and extracellular ActRIIb did not showany role for the final (C-terminal) 15 amino acids (referred to as the“tail” herein) of the extracellular domain in ligand binding. Thissequence failed to resolve on the crystal structure, suggesting thatthese residues are present in a flexible loop that did not packuniformly in the crystal. Thompson et al. EMBO J. 2003 Apr. 1;22(7):1555-66. This sequence is also poorly conserved between ActRIIband ActRIIa. Accordingly, these residues were omitted in the basic, orbackground, ActRIIb-Fc fusion construct. Additionally, position 64 inthe background form is occupied by an alanine, which is generallyconsidered the “wild type” form, although a A64R allele occursnaturally. Thus, the background ActRIIb-Fc fusion has the sequence (Fcportion underlined)(SEQ ID NO:20):

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Surprisingly, the C-terminal tail was found to enhance activin andGDF-11 binding, thus a preferred version of ActRIIb-Fc has a sequence(Fc portion underlined)(SEQ ID NO:21):

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKA variety of ActRIIb a variants that may be used according to themethods described herein are described in the International PatentApplication published as WO2006/012627 (see e.g., pp. 59-60),incorporated herein by reference in its entirety.

Example 6: ActRIIb-hFc Stimulates Erythropoiesis in Non-Human Primates

ActRIIb-hFc (IgG1) was administered once a week for 1-month to male andfemale cynomolgus monkeys by subcutaneous injection. Forty-eightcynomolgus monkeys (24/sex) were assigned to one of four treatmentgroups (6 animals/sex/group) and were administered subcutaneousinjections of either vehicle or ActRIIb-hFc at 3, 10, or 30 mg/kg onceweekly for 4 weeks (total of 5 doses). Parameters evaluated includedgeneral clinical pathology (hematology, clinical chemistry, coagulation,and urinalysis). ActRIIb-hFc caused statistically significant elevatedmean absolute reticulocyte values by day 15 in treated animals. By day36, ActRIIb-hFc caused several hematological changes, including elevatedmean absolute reticulocyte and red blood cell distribution width valuesand lower mean corpuscular hemoglobin concentration. All treated groupsand both sexes were affected. These effects are consistent with apositive effect of ActRIIb-hFc on the release of immature reticulocytesfrom the bone marrow. This effect was reversed after drug was washed outof the treated animals (by study day 56). Accordingly, we conclude thatActRIIb-hFc stimulates erythropoiesis.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

We claim:
 1. A composition for therapeutic administration comprising apolypeptide and a pharmaceutically acceptable carrier, wherein thepolypeptide comprises an amino acid sequence that is at least 95%identical to SEQ ID NO: 2 and wherein the polypeptide promoteserythropoiesis in vivo.
 2. The composition of claim 1, wherein thepolypeptide is at least 98% pure as determined by size exclusionchromatography.
 3. The composition of claim 1, wherein the polypeptidecomprises an amino acid sequence that is at least 97% identical to SEQID NO:
 2. 4. The composition of claim 3, wherein the polypeptidecomprises an amino acid sequence that is at least 99% identical to SEQID NO:
 2. 5. The composition of claim 4, wherein the polypeptidecomprises the amino acid sequence of SEQ ID NO:
 2. 6. The composition ofclaim 1, wherein the polypeptide is glycosylated and has a mammalianglycosylation pattern.
 7. The composition of claim 6, wherein thepolypeptide has a glycosylation pattern obtainable from a chinesehamster ovary (CHO) cell line.
 8. The composition according to claim 1,wherein the N-terminal amino acid is isoleucine.
 9. The composition ofclaim 8, wherein the N-terminus of the polypeptide is ILGRSTQE
 10. Thecomposition of claim 1, wherein the composition is substantially pyrogenfree.
 11. The composition of claim 1, wherein the polypeptide furthercomprises a domain that enhances one or more of in vivo stability, invivo half life, uptake/administration, tissue localization ordistribution, formation of protein complexes, or purification.
 12. Thecomposition of claim 11, wherein the domain is an immunoglobulin Fcdomain.
 13. The composition of claim 11, wherein the domain is serumalbumin.
 14. The composition of claim 1, wherein the serum half life ofthe polypeptide is 20-30 days.
 15. The composition of claim 1, whereinthe polypeptide has one or more of the following characteristics: (i)binds to an ActRIIa ligand with a K_(D) of at least 10⁻⁷ M; and (ii)inhibits ActRIIa signaling.
 16. The composition of claim 1, wherein thepolypeptide is soluble and binds activin A.
 17. The composition of claim16, wherein the polypeptide is an antagonist of activin A.
 18. A methodfor promoting erythropoiesis comprising administering an effectiveamount of the composition of claim
 1. 19. The method according to claim18, wherein the compositions is administered no more frequently thanonce per month.
 20. The method according to claim 19, wherein thecomposition is administered no more frequently than once every twomonths.